title: Identification and characterization of a Mucilaginibacter sp. strain QM49 β-glucosidase and its use in the production of the pharmaceutically active minor ginsenosides (S)-Rh1 and (S)-Rg2.
abstract: Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg1 into (S)-Rg2 and (S)-Rh1, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The Km values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg1 were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the Vmax values were 33.4 ± 0.6 μmol min(-1) mg(-1) of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min(-1) mg(-1) of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh1 and (S)-Rg2 at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh1 and (S)-Rg2 from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Cui
authors.forename: Qing-Mei
authors.initials: QM
authors.lastname: Liu
authors.forename: Jin-Kwang
authors.initials: JK
authors.lastname: Kim
authors.forename: Bong-Hyun
authors.initials: BH
authors.lastname: Sung
authors.forename: Song-Gun
authors.initials: SG
authors.lastname: Kim
authors.forename: Sun-Chang
authors.initials: SC
authors.lastname: Kim
authors.forename: Wan-Taek
authors.initials: WT
authors.lastname: Im
mesh_terms: D041963:Bacteroidetes / Q000201:enzymology* / Q000235:genetics; D001711:Biotransformation; D016000:Cluster Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D036145:Ginsenosides / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D036145:Ginsenosides; D011993:Recombinant Fusion Proteins; D001617:beta-Glucosidase